肌旋毛虫谷氨酰胺酶基因的原核表达及其免疫荧光定位研究
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摘要
采用RT-PCR方法从旋毛虫肌幼虫中扩增出谷氨酰胺酶(TsGls)基因全长序列,将其克隆到表达载体pET-32a(+)中,测序验证后转化到表达宿主菌Rosetta(DE3)pLysS中,用IPTG诱导表达重组蛋白TsGls。以纯化的TsGls蛋白免疫家兔制备多克隆抗体,并对旋毛虫肌幼虫中的TsGls进行免疫荧光定位。结果显示,成功构建了重组质粒pET-32a(+)-TsGls;经SDS-PAGE分析,重组TsGls蛋白的分子质量约为72 ku;制备的多克隆抗体效价可达1.024×10~6;Western-blot分析结果显示,多克隆抗体与重组TsGls具有较强反应性;免疫荧光定位显示,TsGls蛋白存在于旋毛虫肌幼虫皮下组织。上述研究结果为进一步研究旋毛虫谷氨酸依赖型抗酸机制奠定了基础。
To analyze the enzymatic immunogenicity and reactivity of recombinant glutaminese decarboxylase TsGls protein of Trichinella spiralis muscle larvae,the complete sequence of TsGls gene was amplified by RT-PCR,cloned into prokaryotic expression plasmid pET-32a(+),transformed into Rosette(DE3),and then induced with IPTG.The polyclonal antibodies against the recombinant protein was prepared with the purified protein and then the immunofluorescent localization of TsGls in Trichinella spiralis muscle larvae was studied.The recombinant plasmid pET-32a(+)-TsGls was successfully constructed.The recombinant protein r TsGls was confirmed to be about 72 ku in size by SDS-PAGE.The high polyclonal antibody titer was up to 1.024×10~6,indicating good immunogenicity of r TsGls.Western-blot analysis showed high reactivity between r TsGls and larval extracts.Immunofluorescent showed that TsGls protein existed in the subcutaneous tissue of Trichinella spiralis.The above-mentioned results laid foundation for further study of glutamate-dependent acid mechanisms of Trichinella spiralis.
To analyze the enzymatic immunogenicity and reactivity of recombinant glutaminese decarboxylase TsGls protein of Trichinella spiralis muscle larvae,the complete sequence of TsGls gene was amplified by RT-PCR,cloned into prokaryotic expression plasmid pET-32a(+),transformed into Rosette(DE3),and then induced with IPTG.The polyclonal antibodies against the recombinant protein was prepared with the purified protein and then the immunofluorescent localization of TsGls in Trichinella spiralis muscle larvae was studied.The recombinant plasmid pET-32a(+)-TsGls was successfully constructed.The recombinant protein r TsGls was confirmed to be about 72 ku in size by SDS-PAGE.The high polyclonal antibody titer was up to 1.024×10~6,indicating good immunogenicity of r TsGls.Western-blot analysis showed high reactivity between r TsGls and larval extracts.Immunofluorescent showed that TsGls protein existed in the subcutaneous tissue of Trichinella spiralis.The above-mentioned results laid foundation for further study of glutamate-dependent acid mechanisms of Trichinella spiralis.
引文
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[2]徐佳,禹洋,唐颖,等.旋毛虫HSP70基因的原核表达及其免疫原性[J].中国兽医科学,2012,42,(3):275-279.XU Jia,YU Yang,TANG Ying,et al.Prokaryotic expression of HSP70 gene of Trichinella spiralis and immunogenicity analysis of the expression product[J].Chinese Veterinary Science,2012,42(3):275-279.(in Chinese)
[3]GORDEN J,SMALL P L.Acid resistance in enteric bacteria[J].Infect Immun,1993,61(1):364-367.
[4]SMALL P,BLANKENHORN D,WELTY D,et al.Acid and base resistance in Escherichia coli and Shigella flexneri:role of rpo S and growth p H[J].J Bacteriol,1994,176(6):1729-1737.
[5]EGUCHI Y,UTSUMI R.Introduction to bacterial signal transduction networks[J].Adv Exp Med Biol,2008,63(1):1-6.
[6]TUCKER D L,TUKER N,CONWAY T.Gene expression profiling of the p H response in Escherichia coli[J].J Bacteriol,2002,184(23):6551-6558.
[7]LU P,MA D,CHEN Y,et al.L-glutamine provides acid resistance for Escherichia coli through enzymatic release of ammonia[J].Cell Res,2013,23(5):635-644.
[8]LI F,CUI J,WANG Z Q,et al.Sensitivity and optimization of artificial digestion in the inspection of meat for Trichinella spiralis[J].Foodborne Pathog Dis,2010,7(8):879-885.
[9]高慎阳,查恩辉,王珅,等.一种“高性价比”切胶纯化原核表达蛋白的方法[J].中国农学通报,2010,26(22):24-26.GAO Shen-yang,ZHA En-hui,WANG Shen,et al.A'Cost-effective'method for purification of prokaryotic expression proteins in gelslices[J].Chinese Agricultural Science Bulletin,2010,269(22):24-26.(in Chinese)
[10]周兴东,王泽,宋铭忻,等.旋毛虫谷氨酸脱羧酶基因的原核表达及其活性研究[J].中国兽医科学,2015;45(2):132-136.ZHOU Xing-dong,WANG Ze,SONG Ming-xin,et al.Prokaryotic expression of Trichinella spiralis glutamate decarboxylase and activity characteristic analysis of the expressed protein[J].Chinese Veterinary Science,2015;45(2):132-136.(in Chinese)
[11]CASTANIE-CORNET M P,PENFOUND T A,SMITH D,et al.Control of acid resistance in Escherichia coli[J].J Bacteriol,1999,181(11):3525-3535.
[12]MA D,LU P,YAN C,et al.Structure and mechanism of a glutamate-GABA antiporter[J].Nature,2012,483(7391):632-636.
[13]MITREVA M,JASMER D P,ZARLENGA D S,et al.The draft genome of the parasitic nematode Trichinella spiralis[J].Nat Genet,2011,43(3):228-235.